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Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells.

Cattaneo L, Cicconi R, Mignogna G, Giorgi A, Mattei M, Graziani G, Ferracane R, Grosso A, Aducci P, Schininà ME, Marra M - PLoS ONE (2015)

Bottom Line: Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action.Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays.Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of "Tor Vergata", Rome, Italy.

ABSTRACT
Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

No MeSH data available.


Related in: MedlinePlus

Representative 2-DE map of control A375 melanoma cells.Proteins were electrophoretically separated in the non-linear pH range 3–10 and the 200–15 kDa molecular mass range and visualized by colloidal Coomassie staining. The encircled spots indicate the proteins affected by rosemary extract (1:240; 24h) treatment. The relative intensities, between control and treated samples, are shown in panels. Data significance was evaluated by a Student’s t-test (P ≤0.05). PDA3, protein disulfide-isomerase A3; GANAB, Neutral alpha-glucosidase AB; PCBP1, poly(rC)- binding protein 1; PCBP2, poly(rC)-binding protein 2; LMNA, lamin A.
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pone.0132439.g006: Representative 2-DE map of control A375 melanoma cells.Proteins were electrophoretically separated in the non-linear pH range 3–10 and the 200–15 kDa molecular mass range and visualized by colloidal Coomassie staining. The encircled spots indicate the proteins affected by rosemary extract (1:240; 24h) treatment. The relative intensities, between control and treated samples, are shown in panels. Data significance was evaluated by a Student’s t-test (P ≤0.05). PDA3, protein disulfide-isomerase A3; GANAB, Neutral alpha-glucosidase AB; PCBP1, poly(rC)- binding protein 1; PCBP2, poly(rC)-binding protein 2; LMNA, lamin A.

Mentions: In order to get hints about the molecular mechanism underlying rosemary extract cytotoxicity, a proteomic analysis was carried out, to ascertain qualitative and/or quantitative modification of the protein profile of melanoma cells subjected to rosemary extract treatment, as compared to control cells. To this purpose, total proteins were extracted from cells treated with 1:240 dilution of rosemary extract for 24 h, or from untreated cells and resolved by two dimensional gel electrophoresis (2-DE). To detect quantitative changes in relative spot volumes of proteins from treated cells as compared to control ones, colloidal Coomassie-stained gels were subjected to software-assisted image analysis. Statistical evaluation of the relative spot volumes allowed to detect spots significantly varying (P ≤ 0.05) in abundance. Representative 2-D gel is shown in Fig 6. The overall 2-DE profiles of control and treated cells were similar, however, 5 protein spots were detected, whose abundance was at least two-fold reduced in response to rosemary challenge; no proteins with a corresponding increase were detected. The differential spots were excised from the gel, proteolysed and subjected to MS analysis. The database search with results from Peptide Mass Fingerprinting MALDI-ToF experiments, allowed the identification of protein spots. The list of the identified polypeptides is reported in Table 2. All reported proteins had good sequence coverage, significant protein scores, as well as similar experimental and theoretical Mw/pI.


Anti-Proliferative Effect of Rosmarinus officinalis L. Extract on Human Melanoma A375 Cells.

Cattaneo L, Cicconi R, Mignogna G, Giorgi A, Mattei M, Graziani G, Ferracane R, Grosso A, Aducci P, Schininà ME, Marra M - PLoS ONE (2015)

Representative 2-DE map of control A375 melanoma cells.Proteins were electrophoretically separated in the non-linear pH range 3–10 and the 200–15 kDa molecular mass range and visualized by colloidal Coomassie staining. The encircled spots indicate the proteins affected by rosemary extract (1:240; 24h) treatment. The relative intensities, between control and treated samples, are shown in panels. Data significance was evaluated by a Student’s t-test (P ≤0.05). PDA3, protein disulfide-isomerase A3; GANAB, Neutral alpha-glucosidase AB; PCBP1, poly(rC)- binding protein 1; PCBP2, poly(rC)-binding protein 2; LMNA, lamin A.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4503536&req=5

pone.0132439.g006: Representative 2-DE map of control A375 melanoma cells.Proteins were electrophoretically separated in the non-linear pH range 3–10 and the 200–15 kDa molecular mass range and visualized by colloidal Coomassie staining. The encircled spots indicate the proteins affected by rosemary extract (1:240; 24h) treatment. The relative intensities, between control and treated samples, are shown in panels. Data significance was evaluated by a Student’s t-test (P ≤0.05). PDA3, protein disulfide-isomerase A3; GANAB, Neutral alpha-glucosidase AB; PCBP1, poly(rC)- binding protein 1; PCBP2, poly(rC)-binding protein 2; LMNA, lamin A.
Mentions: In order to get hints about the molecular mechanism underlying rosemary extract cytotoxicity, a proteomic analysis was carried out, to ascertain qualitative and/or quantitative modification of the protein profile of melanoma cells subjected to rosemary extract treatment, as compared to control cells. To this purpose, total proteins were extracted from cells treated with 1:240 dilution of rosemary extract for 24 h, or from untreated cells and resolved by two dimensional gel electrophoresis (2-DE). To detect quantitative changes in relative spot volumes of proteins from treated cells as compared to control ones, colloidal Coomassie-stained gels were subjected to software-assisted image analysis. Statistical evaluation of the relative spot volumes allowed to detect spots significantly varying (P ≤ 0.05) in abundance. Representative 2-D gel is shown in Fig 6. The overall 2-DE profiles of control and treated cells were similar, however, 5 protein spots were detected, whose abundance was at least two-fold reduced in response to rosemary challenge; no proteins with a corresponding increase were detected. The differential spots were excised from the gel, proteolysed and subjected to MS analysis. The database search with results from Peptide Mass Fingerprinting MALDI-ToF experiments, allowed the identification of protein spots. The list of the identified polypeptides is reported in Table 2. All reported proteins had good sequence coverage, significant protein scores, as well as similar experimental and theoretical Mw/pI.

Bottom Line: Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action.Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays.Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of "Tor Vergata", Rome, Italy.

ABSTRACT
Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed.

No MeSH data available.


Related in: MedlinePlus